GSM7712989: TEinsertion,normal,RNA,rep3; Zea mays; RNA-Seq
Total RNA from the maize leaf samples was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, United States). Total amounts and integrity of RNA were assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). The Halo-ZmCCT or Halo-ZmAux/IAA8 fusion protein was expressed using the TNT SP6 Coupled Wheat Germ Extract System (Promega, USA) and then purified with Magne HaloTag Beads (Promega, USA). Genomic DNA (gDNA, 5 μg in 130 μL TE buffer) was fragmented to an average of 200 bp using a Covaris M220 (Woburn, USA) according to the manufacturer's recommended settings. The fragmented gDNA was then purified using MICH DNA Clean Beads (Bluescape, China) at a DNA to beads ratio of 0.7-1.1. The beads were incubated with the gDNA for 5 min at room temperature and placed on a magnet (Bluescape, China) to immobilize the beads. The supernatant was then removed. The beads were washed twice with 200 μL of 80% ethanol and allowed to dry. Once dry, they were resuspended in 22 μL resuspension buffer, incubated at room temperature for 5 min, and placed on the magnet. The DNA-containing supernatant was then transferred to a new tube. Briefly, mRNA was purified by using poly-T oligo-attached magnetic beads. First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase. The library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then, the PCR product was purified by AMPure XP beads, and the library was finally obtained. DAP-seq libraries were constructed using the MICH TLX DNA-seq Kit (Bluescape, China) according to the manufacturer's instructions. After the beads with bound proteins were incubated with DNA fragments at room temperature for 1 h, they were washed three times with wash buffer to remove unbound DNA fragments. The HaloTag beads were resuspended in 30-μl elution buffer and then heated at 98 °C for 10 min to denature the protein and release the bound DNA fragment into the solution.